Therapeutic Compositions

ABSTRACT

The invention relates to a product containing the compound of formula (I) below (I) or a pharmaceutically acceptable salt of this compound, in combination with at least one compound having PDE5-inhibitory properties, or a pharmaceutically acceptable salt thereof, for therapeutic use, simultaneously, separately or over a period of time, in the treatment of a disease wherein vasoconstriction is involved.

This application is a continuation of U.S. application Ser. No.16/942,895, filed Jul. 30, 2020, which is a continuation of U.S.application Ser. No. 16/599,582, filed Oct. 11, 2019, which is acontinuation of U.S. application Ser. No. 16/193,859, filed Nov. 16,2018, which is a continuation of U.S. application Ser. No. 15/881,060,filed Jan. 26, 2018, which is a continuation of U.S. application Ser.No. 13/604,148, filed Sep. 5, 2012, which is a continuation of U.S.application Ser. No. 12/439,290, filed Feb. 27, 2009, which is a USnational stage of International Appl. No. PCT/IB2007/053448, filed Aug.28, 2007, which claims priority to International Appl. No.PCT/IB2006/053857, filed Oct. 19, 2006 and International Appl. No.PCT/IB2006/052999, filed Aug. 29, 2006, the disclosures of which areincorporated herein by reference in their entireties.

The present invention relates to a product containing the compound offormula (I) below

or a pharmaceutically acceptable salt of this compound, in combinationwith at least one compound having PDE5-inhibitory properties, or apharmaceutically acceptable salt thereof, for therapeutic use,simultaneously, separately or over a period of time, in the treatment ofa disease wherein vasoconstriction is involved.

PCT publication WO 02/053557 describes endothelin receptor antagonistsincluding the compound of formula (I) and the use of said endothelinreceptor antagonists in the treatment of various diseases whereinvasoconstriction is involved (i.a. heart failure, angina pectoris,pulmonary and systemic hypertension and erectile dysfunction).

PDE-5 inhibitors have been notably described in the following patentdocuments:

-   -   U.S. Pat. No. 5,250,534 (describing pyrazolopyrimidinone        derivatives as PDE-5 inhibitors and i.a. sildenafil as well as        the use of the same for i.a. hypertension and heart failure) and        EP 1 097 711 (describing i.a. sildenafil for pulmonary        hypertension);    -   WO 99/24433 (describing i.a. vardenafil as well as the use of        the same for i.a. hypertension, angina pectoris and erectile        dysfunction);    -   U.S. Pat. No. 5,859,006 (describing i.a. tadalafil as well as        the use of the same for i.a. hypertension, pulmonary        hypertension, angina and congestive heart failure);    -   WO 00/27848 (describing i.a. udenafil and the use thereof for        impotence).

The Applicant has now surprisingly found that the combination of thecompound of formula (I) with a compound having PDE5-inhibitoryproperties results in an unexpected synergistic effect in the treatmentof diseases wherein vasoconstriction is involved.

Therefore a subject of this invention is a product containing thecompound of formula (I) as described before or a pharmaceuticallyacceptable salt of said compound of formula (I), in combination with atleast one (and preferably one) compound having PDE5-inhibitoryproperties, or a pharmaceutically acceptable salt thereof, fortherapeutic use, simultaneously, separately or over a period of time, inthe treatment of a disease wherein vasoconstriction is involved.

The following paragraphs provide definitions of the various terms usedin the present patent application and are intended to apply uniformlythroughout the specification and claims, unless an otherwise expresslyset out definition provides a broader or narrower definition.

“PDE-5” stands in the present application for cyclic guanosome 3′,5′-monophosphate (cGMP) phosphodiesterase type 5.

“Simultaneously” or “simultaneous”, when referring to a therapeutic use,means in the present application that the therapeutic use concernedconsists in the administration of two or more active ingredients by thesame route and at the same time.

“Separately” or “separate”, when referring to a therapeutic use, meansin the present application that the therapeutic use concerned consistsin the administration of two or more active ingredients at approximatelythe same time by at least two different routes.

By therapeutic administration “over a period of time” is meant in thepresent application the administration of two or more ingredients atdifferent times, and in particular an administration method according towhich the entire administration of one of the active ingredients iscompleted before the administration of the other or others begins. Inthis way it is possible to administer one of the active ingredients forseveral months before administering the other active ingredient oringredients. In this case, no simultaneous administration occurs.

By “disease wherein vasoconstriction is involved” is meant in particularhypertension, pulmonary hypertension (including pulmonary arterialhypertension), diabetic arteriopathy, heart failure, erectiledysfunction or angina pectoris.

By “compound having PDE5-inhibitory properties” is meant a compoundthat, when submitted to the “Test for the determination of PDE5 IC₅₀”described in the present patent application, has an IC₅₀ equal or lowerthan 1 μM.

Specific examples of compounds having PDE5-inhibitory properties includethe compounds having the following structures (names):

The term “pharmaceutically acceptable salts” refers to non-toxic,inorganic or organic acid and/or base addition salts. Reference can bemade to “Salt selection for basic drugs”, Int. J. Pharm. (1986), 33,201-217.

Besides, any reference to a compound of formula (I) or to a compoundhaving PDE5-inhibitory properties is to be understood as referring alsoto the pharmaceutically acceptable salts thereof, as appropriate andexpedient.

Preferably, the product according to this invention will be such thatthe compound of formula (I) and the compound having PDE5-inhibitoryproperties are intended for a therapeutic use which takes placesimultaneously or over a period of time.

According to one preferred variant of this invention, the compound offormula (I) and the compound having PDE5-inhibitory properties will beintended to be administered simultaneously.

According to another preferred variant of this invention, the compoundof formula (I) and the compound having PDE5-inhibitory properties willbe intended to be administered over a period of time.

The period of time intended for the therapeutic use of a productaccording to this invention will be at least one week, and preferably atleast one or more months (for example six months). This period of timemay also be the whole life of the patient that receives the product.Preferably, administration of compound of formula (I) will be alternatedwith administration of a compound having PDE5-inhibitory properties, andthe interval between such administration will not exceed two or threedays (and more preferably not exceed one day).

Preferably, the compound having PDE5-inhibitory properties will beselected from sildenafil, vardenafil, tadalafil and udenafil. Morepreferably, the compound having PDE5-inhibitory properties will besildenafil or tadalafil.

According to one particularly preferred variant of the invention thecompound having PDE5-inhibitory properties will be sildenafil.

According to another particularly preferred variant of the invention thecompound having PDE5-inhibitory properties will be tadalafil.

The administration route of the compound of formula (I) and that of thecompound having PDE5-inhibitory properties is preferably the same. Inparticular, the common administration route for the compound of formula(I) and for the compound having PDE-inhibitory properties will be theintravenous or oral route (and notably the oral route).

Though the exact administration doses of a product according to thisinvention will have to be determined by the treating physician, it isexpected that a dose of 0.05 to 2 mg (and preferably 0.1 to 1 mg) ofcompound of formula (I) per kg of patient body weight combined with adose 0.01 to I mg (and preferably 0.05 to 0.5 mg) of tadalafil per kg ofpatient body weight, both compounds being intended to be administered byoral route, or combined with a dose 0.1 to 2 mg (and preferably 0.2 to 1mg) of sildenafil per kg of patient body weight will be appropriate,both compounds being intended to be administered by oral route as well.

Preferably, the disease intended to be treated by a product according tothis invention will be selected from hypertension, pulmonaryhypertension, diabetic arteriopathy, heart failure, erectile dysfunctionand angina pectoris. More preferably, the disease intended to be treatedby a product according to this invention will be selected fromhypertension and pulmonary hypertension. In particular, the diseaseintended to be treated by a product according to this invention will bepulmonary hypertension (and notably pulmonary arterial hypertension).

The invention also relates to a pharmaceutical composition containing,as active principles, the compound of formula (I) below

or a pharmaceutically acceptable salt of this compound, in combinationwith at least one (and preferably one) compound having PDE5-inhibitoryproperties, or a pharmaceutically acceptable salt thereof, as well as atleast one excipient.

The invention further relates to the use of the compound of formula (I)below

or a pharmaceutically acceptable salt of this compound, in combinationwith at least one (and preferably one) compound having PDE5-inhibitoryproperties, or a pharmaceutically acceptable salt thereof, for themanufacture of a medicament intended to treat a disease whereinvasodilation is required.

Besides, preferences indicated for the product according to thisinvention of course apply mutatis mutandis to the pharmaceuticalcompositions and uses of this invention.

Particular embodiments of the invention are described in the followingExamples, which serve to illustrate the invention in more detail withoutlimiting its scope in any way.

EXAMPLES

To illustrate the usefulness of this invention, the association of thecompound of formula (I), administered orally at a dose of 0.3 mg/kg,with tadalafil, administered orally at a dose of 10 mg/kg, has beenstudied in two different hypertension models, namely the Dahlsalt-sensitive rat model and the spontaneously hypertensive rat model.Furthermore, the association of the compound of formula (I),administered orally at a dose of 0.3 mg/kg, with sildenafil,administered orally at a dose of 30 mg/kg, has been studied in thespontaneously hypertensive rat model. The protocols used are detailed inthe part entitled “Pharmacological properties of the inventioncompounds” hereafter.

Pharmacological Properties of the Invention Compounds

The experimental methods described hereafter can be used to show thepharmacological properties of the invention compounds.

Dahl Salt-Sensitive Rat Model

Dahl salt-sensitive (Dahl-S) were purchased from Harlan (Netherlands).The rats were housed by group during the acclimatization period andsingle-housed after implantation of the telemetry device. All animalswere maintained under identical conditions and had free access to normalpelleted rat chow and water. Dahl salt-sensitive rats develophypertension only upon exposure to salt intake. They were administered ahigh-salt (8%) chow diet (Purina series 5500). Five weeks after startingsalt administration, a telemetry system was implanted under anaesthesiaby inhalation of 2.5% isoflurane (in 70% O₂+30% N₂O). Under asepticconditions, a pressure radio-frequency transmitter was implanted intothe peritoneal cavity, and a sensing catheter was inserted in thedescending aorta and advanced pointing upstream to slightly below therenal artery bifurcation. The transmitter was sutured to the abdominalmusculature and the skin was closed. A receiver platform transformed theradio signal into digitized input, that was sent to a dedicated personalcomputer (Compaq, deskpro). Arterial blood pressure measurements werecalibrated by using an input from an ambient pressure reference.Telemetry units were obtained from Data Sciences (St. Paul, Minn., USA).

The compounds were administered at least 2 weeks after telemetry systemimplantation. The compound of formula (I) and the compound havingPDE5-inhibitory properties were prepared in 5% arabic gum andadministered by oral gavage. The acute effects of the compound offormula (l), the compound having PDE5-inhibitory properties and theircombination on blood pressure were measured by collecting data at5-minute intervals up to 72 h after oral administration. Hourly means ofblood pressure were calculated for each rat. Each rat served as its owncontrol, by using the blood pressure data of the last 24 hours beforedrug administration. The two curves (blood pressure of the controlperiod and blood pressure of the treatment period) were plotted togetherand the area between curves (ABC) from 0 to 72 hours was calculated. Thehigher the ABC, the stronger the effect of the item tested for loweringblood pressure.

Spontaneously Hypertensive Rat Model

The same protocol was used as for the Dahl salt-sensitive rat model,except that spontaneously hypertensive rats (SHR) replace the Dahl-Srats and that the SHR rats did not receive any salt dict. The SHR ratswere purchased from Harlan (Netherlands).

Test for the Determination of PDE5 IC₅₀:

In order to estimate the extent of inhibition for PDE5 activity of atest compound, the following test is carried out. Phosphodiesterase-5enzyme (PDE 5) is separated from human corpus cavernosal tissues. About3 g of this tissue is homogenized with 12 ml of HEPES buffer (20 mMHEPES, 250 mM sucrose, 1 mM EDTA, 1 mM PMSF, pH 7.2) at 4° C. Thesolution is filtered with double-layered gauze and centrifuged(100,000×g) for 60 min at 4° C. The supernatant is filtered with 0.2 μmfilter paper and separated by HPLC (Mono Q anion exchange column) withconcentration gradient of 0-500 mM NaCl to elute PDE isozymes. Theenzyme activity is measured on each column fraction by the followingprocess to separate the PDE5 fraction and the PDE5 inhibition of thetest compound is measured using the PDE5 fraction. To 1.5 ml tube areadded 100 μl of reaction mixture (15 mM Tris-HCl, 5 mM MgCl₂, 0.5 mg/mlBSA, pH 7.4) and the appropriate amount of test compound fraction andtest compound and the mixture is mixed well. To this solution is added³H-cAMP or ³H-cGMP (500 nM, 2 μCi/ml), the mixture is reacted in theincubator of 30° C. for about 1 hour and the reaction is quenched byputting the tube into boiling water for about 45 seconds to 2 min. Thenthe tube is chilled in ice bath for about 5 min. To this tube is addedsnake venom (1 mg/ml, 100 μl) or 5-nucleotidase (0.1 unit/tube) and themixture is reacted in an incubator at 37° C. for 10 min and chilled inan ice bath. 3 times volume of methanol to the resin is added to theanion exchange resin (Bio-Rad resin, AG1-X2, 200-400 mesh) which hasbeen already washed with 0.5N HCl, H₂O, 0.5N NaOH, H₂O, 0.5N HCl and H₂Oin order and adjusted to pH 5. Then 1 ml of the pretreated resin isdispensed into each tube with vortexing. The mixture is left at 4° C.for 15 min with occasional vortexing and centrifuged (10.000 rpm) forabout 5 min to sediment the resin. The supernatant (700 μl) istransferred to a liquid scintillation vial, and mixed with 10 ml ofscintillation cocktail. After stabilizing the solution by leaving itovernight, the radioactivity of the tube is measured by β-counter.

If the test compound has an IC₅₀ equal or lower than 1 μM, it isconsidered as having PDE5-inhibitory properties for the purpose of thispatent application. If the test compound has an IC₅₀ higher than 1 μM,it is considered as not having PDE5-inhibitory properties for thepurpose of this patent application.

Example 1

Following the test protocol described previously in the part entitled“Dahl salt-sensitive rat model”, oral administration of the compound offormula (I) and tadalafil decreased blood pressure in Dahl-S rats: thecompound of formula (I) (0.3 mg/kg) decreased blood pressure with an ABCof 256 and tadalafil (10 mg/kg) with an ABC of 310. The ABC after oraladministration of the combination (compound of formula (I) at 0.3 mg/kgand tadalafil at 10 mg/kg) was 923, demonstrating a synergistic effect.

Example 2

Following the test protocol described previously in the part entitled“Spontaneously hypertensive rat model”, oral administration of thecompound of formula (I) and tadalafil decreased blood pressure in SHRrats: the compound of formula (I) (0.3 mg/kg) decreased blood pressurewith an ABC of 44 and tadalafil (10 mg/kg) with an ABC of 286. The ABCafter oral gavage of the combination (compound of formula (I) at 0.3mg/kg and tadalafil at 10 mg/kg) was 444, confirming the synergisticeffect.

Example 3

Following the test protocol described previously in the part entitled“Spontaneously hypertensive rat model”, oral administration of thecompound of formula (I) and sildenafil decreased blood pressure in SHRrats: the compound of formula (I) (0.3 mg/kg) decreased blood pressurewith an ABC of 38 and sildenafil (30 mg/kg) with an ABC of 229. The ABCafter oral gavage of the combination (compound of formula (I) at 0.3mg/kg and sildenafil at 30 mg/kg) was 317, confirming the synergisticeffect.

What is claimed is:
 1. A method for the treatment of pulmonary arterialhypertension comprising administering simultaneously, separately, orover a period of time, to a patient in need thereof, an effective amountof a compound of formula (I):

in free or pharmaceutically acceptable salt form, and at least onecompound having PDE5-inhibitory properties, in free or pharmaceuticallyacceptable salt form.
 2. The method of claim 1, wherein the compound offormula (I) and the compound having PDE5-inhibitory properties areadministered simultaneously.
 3. The method of claim 1, wherein thecompound of formula (I) and the compound having PDE5-inhibitoryproperties are administered separately.
 4. The method of claim 1,wherein the compound of formula (I) and the compound havingPDE5-inhibitory properties are administered over a period of time. 5.The method of claim 4, wherein the interval between the administrationof the compound of formula (I) and the administration of the compoundhaving PDE5-inhibitory properties does not exceed two days.
 6. Themethod of claim 5, wherein the interval does not exceed one day.
 7. Themethod of claim 1, wherein the compound having PDE5-inhibitoryproperties is sildenafil, vardenafil, tadalafil, or udenafil.
 8. Themethod of claim 1 wherein the compound having PDE5-inhibitory propertiesis sildenafil.
 9. The method of claim 1, wherein the compound havingPDE5-inhibitory properties is tadalafil.